Sequence specific antimicrobials

Active Publication Date: 2016-01-28
THE ROCKEFELLER UNIV
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  • Abstract
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Benefits of technology

[0006]In another aspect the disclosure provides methods for reducing the amount of virulent and/or antibiotic resistant bacteria or plasmids in a bacterial population. The method comprises contacting the bacterial population with a pharmaceutical composition as described herein, wherein contacting the bacterial population is such that at least some of the phagemids are introduced into at least some of the bacteria in the bacterial population and, subsequent to the introduction of the phagemids, at least the targeting RNA and the Cas enzyme are produced. Subsequent to production of at least the targeting RNA and the Cas enzyme, the amount of unwanted bacteria in the population, such as the amount of virulent and/or antibiotic resistant bacteria, or plasmids in the mixed bacterial population, is reduced.
[0007]In another aspect, the disclosure includes a method for personalized therapy for an individual in need of therapy for a bacterial infection. The method comprises obtaining a biological sample from the individual and determining a plurality of bacterial DNA sequences from the sample. The biological sample can be any biological sample, including but not necessarily limited to a liquid biological sample, such as blood, mucous, serum, cerebrospinal fluid, saliva, and urine, or a solid biological sample, such as a biopsy of any tissue, or it can comprise a sample obtained by using an im

Problems solved by technology

The study of the microbiome not only shows the importance of certain species for the human host, but has also revealed the undesired side-effects of traditio

Method used

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example 1

[0052]In the present disclosure we demonstrate the feasibility of using packaged phagemids encoding CRISPR systems as sequence-specific antimicrobials that facilitate the selective killing bacteria within a heterogeneous bacteria population. To demonstrate specific embodiments of this approach, we used the cas9 gene and its RNA guide / s sequences using a phagemid, a plasmid designed to be packaged in phage capsids (FIG. 1A). We demonstrate the general applicability of this approach by using Staphylococcus aureus. We show that we can selectively kill antibiotic-resistant and / or virulent Staphylococcus aureus strains which for a variety of well-known reasons are pertinent to public health. In particular, staphylococci are at the same time the predominant members of the human skin microbiota and one of the most common causes of nosocomial infections. The recent increase in staphylococcal pathogenicity is largely the result of the transfer of antibiotic resistance and virulence genes via...

example 2

[0053]In order to develop a phagemid system for Staphylococcus aureus, we cloned a ˜2 kb fragment containing the rinA, terS and terL genes and packaging site from the staphylococcal ΦNM1 phage on plasmid pC194, obtaining pDB91. To assess the efficiency of packaging of pDB91 in ΦNM1 capsids, a transduction assay was performed. RN4220 cells containing pDB91 were infected with ΦNM1, and the lysate was used to transduce RN4220 cells previously lysogenized with ΦNM1, referred to as RNΦ. The lysogenic strain is resistant to superinfection which allows observation of phagemid transduction while negating the presence of wild-type phage in the lysate. We determined that a lysate with a titer of 1.6×107 plaque forming units (PFU) / μl contained 3.8×106 transfer units (TU) / μl, suggesting both a high efficiency of phagemid packaging (TU / PFU, 24%) as well as a high TU titer to treat a large bacterial population (FIG. 5). We cloned the CRISPR sequences of pDB114 and pDB144::aph into the pDB91 phage...

example 3

[0054]Following treatment with conventional antibiotics that eradicate most members of a bacterial community, the rise and propagation of resistance is fueled by the lack of competitors in the treated environment. As shown above, sequence-specific killing is not exempt from the generation of cells that escape treatment, however this strategy has the benefit of killing only a subpopulation, leaving other members of the bacterial community and even of the same species to colonize the niche and limit the propagation of resistant organisms. To demonstrate this effect, we performed a treatment of RNKΦ in which the pCN57 GFP reporter plasmid was transformed, either alone or in a co-culture with RNΦ. In both cases, the treatment effectively kills the targeted population, as evidenced by an interrupted increase of OD in the monoculture and GFP signal in the mixed culture respectively (FIG. 1D). After 7 hours, RNKΦ cells that survived the treatment resume growth in the mono-culture whereas t...

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Abstract

Provided are compositions and methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population, or for reducing any other type of unwanted bacteria in a mixed bacteria population. The compositions and methods involve targeting bacteria that are differentiated from other members of the population by at least one unique clustered regularly interspaced short palindromic repeats (CRISPR) targeted DNA sequence. The compositions and methods can be readily adapted to target any bacteria or any bacteria plasmid, or both.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application No. 61 / 761,971, filed on Feb. 7, 2013, the disclosure of which is incorporated herein by reference.FIELD OF THE DISCLOSURE[0002]The present disclosure is related to compositions and methods for selectively killing specific bacteria in mixed bacteria populations.BACKGROUND OF THE DISCLOSURE[0003]Advances in DNA sequencing technologies have revealed the diversity of complex microbial populations in many different environments. They have also provided evidence for the contributions that individual species make to the whole of the population and / or to their environment. Perhaps the most striking example of this is the human microbiome and its influence on human health. The study of the microbiome not only shows the importance of certain species for the human host, but has also revealed the undesired side-effects of traditional antimicrobials without killing specificity, which in...

Claims

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Application Information

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IPC IPC(8): C12N15/74A01N63/00A61K38/46C12N9/22
CPCC12N15/74A01N63/00C12Y301/00C12N9/22A61K38/465C12N9/16A61K45/06A61K31/7105A61K31/713C12N15/113C12N2310/10C12N2310/20A61K2300/00C12N2795/10331C12N2795/10332C12N2795/10343C12N2795/10371
Inventor BIKARD, DAVIDMARRAFFINI, LUCIANO
Owner THE ROCKEFELLER UNIV
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